Quantitative Characterization of Local Protein Solvation To Predict Solvent Effects on Protein Structure

Characterization of solvent preferences of proteins is essential to the understanding of solvent effects on protein structure and stability. Although it is generally believed that solvent preferences at distinct loci of a protein surface may differ, quantitative characterization of local protein solvation has remained elusive. In this study, we show that local solvation preferences can be quantified over the entire protein surface from extended molecular dynamics simulations. By subjecting microsecond trajectories of two proteins (lysozyme and antibody fragment D1.3) in 4 M glycerol to rigorous statistical analyses, solvent preferences of individual protein residues are quantified by local preferential interaction coefficients. Local solvent preferences for glycerol vary widely from residue to residue and may change as a result of protein side-chain motions that are slower than the longest intrinsic solvation timescale of ～10 ns. Differences of local solvent preferences between distinct protein side-chain conformations predict solvent effects on local protein structure in good agreement with experiment. This study extends the application scope of preferential interaction theory and enables molecular understanding of solvent effects on protein structure through comprehensive characterization of local protein solvation.     

Non-polar solutes in water and in aqueous solutions of protein denaturants. Modeling of solution and transfer processes

A simple molecular model for the thermodynamic behavior of non-polar solutes in water and in aqueous solutions of protein denaturants is presented. Three contributions are considered: (i) combinatorial arising from the mixing process, (ii) interactional characterizing the molecular interactions occurring in the mixture and (iii) a contribution originating from the structural changes occurring in the first shell of water molecules around the solute. The latter is modeled assuming that water molecules in contact with the solute are involved in a chemical equilibrium between two states. The model describes well the temperature and denaturant concentration dependences of the Gibbs energies of solution and transfer for benzene, toluene and alkanes in water and aqueous solutions of urea and guanidine hydrochloride. Model parameters are physically meaningful, allowing a discussion of the molecular interactions involved. A preferential solvation of the solute by the denaturant is found. However, the non-polar solute-denaturant interaction is not specific, i.e. leading to a distinct chemical entity. Urea and guanidine hydrochloride are non-polar solubilizing agents because their interactions with the solute are less unfavorable than those between water and the solute.     

Preferential interaction of dimethyl sulfoxide and phosphatidyl choline membranes

The interaction free energy of dimethyl sulfoxide (DMSO) and two types phospholipid membranes has been assessed from measurements of vapor pressure. The lipids were phosphatidyl cholines with respectively (14:0/14:0) (DMPC) and (16:0/18:1) (POPC) fatty acid chains. The results were expressed in terms of the iso-osmolal preferential interaction parameter, Γ_μ1, which remained negative under all experimental conditions investigated here. This shows that water-membrane interactions are more favorable than DMSO-membrane interactions. This condition is known as preferential exclusion of DMSO (or preferential hydration of the membrane), and implies that the local (interfacial) concentration of the solute is reduced compared to the bulk. At room temperature and 1 m DMSO, Γ_μ1 was −0.3 to −0.4 for both lipids. This corresponds to a sizable reduction in the DMSO concentration in a zone including at least the first two hydration layers of the membrane. Possible origins of the preferential exclusion are discussed.As a direct consequence of the pronounced preferential exclusion, DMSO generates an osmotic stress at the membrane interface. This tends to stabilize lipid phases of low surface areas and to withdraw water from multilamellar stacks of membranes. Based on this, we suggest that the preferential exclusion of DMSO explains both the modulation of phase behavior and the constriction of multilamellar aggregates induced by this solute.     

Simulation of peptide folding with explicit water--a mean solvation method

A new approach to efficiently calculate solvent effect in computer simulation of macromolecular systems has been developed. Explicit solvent molecules are included in the simulation to provide a mean solvation force for the solute conformational search. Simulations of an alanine dipeptide in aqueous solution showed that the new approach is significantly more efficient than conventional molecular dynamics method in conformational search, mainly because the mean solvation force reduced the solvent damping effect. This approach allows the solute and solvent to be simulated separately with different methods. For the macromolecule, the rigid fragment constraint dynamics method we developed previously allows large time-steps. For the solvent, a combination of a modified force-bias Monte Carlo method and a preferential sampling can efficiently sample the conformational space. A folding simulation of a 16-residue peptide in water showed high efficiency of the new approach.     

A CFF 91-based Continuum Solvation Model: Solvation Free Energies of Small Organic Molecules and Conformations of the Alanine Dipeptide in Solution

Abstract

For molecular mechanics simulations of solvated molecules, it is important to use a consistent approach for calculating both the force field energy and the solvation free energy. A continuum solvation model based upon the atomic charges provided with the CFF91 force field is derived. The electrostatic component of the solvation free energy is described by the Poisson-Bolzmann equation while the nonpolar comonent of the solvation energy is assumed to be proportional to the solvent accessible surface area of the solute. Solute atomic radii used to describe the interface between the solute and solvent are fitted to reproduce the energies of small organic molecules. Data for 140 compounds are presented and compared to experiment and to the results from the well-characterized quantum mechanical solvation model AM1-SM2. In particular, accurate results are obtained for amino acid neutral analogues (mean unsigned error of 0.3 kcal/mol). The conformational energetics of the solvated alanine dipeptide is discussed.     

The effects of solvent on dioxygen binding to cobalt(II) picket fence porphyrins

The O₂ affinities on base adducts of four atropisomers of picket fence porphyrin Co(TpivPP), and the corresponding α⁴ complex containing valeramido pickets instead of pivalamido Co(α⁴-TvalPP) were measured in several solvents at O or −15 °C. The O₂ affinities of the α⁴ complexes are the lowest in DMF which is the most polar of the solvents used, while those of the other isomers are the highest in DMF. This observation was explained in terms of direct and indirect interactions between the solvent and the bound O₂. The trans-α² complex shows higher O₂ affinity in dichloromethane than those in aromatic solvents because of the preferential solvation of the deoxy complex in the solvents. The variation of the O₂ affinities of this system to solvents is considerably smaller than those of ‘flat porphyrin’ complexes. This result suggested that the pocket polarity introduced by the amide groups weakens the solvent–solute interaction on the O₂ affinities of this system and also that the solvation of the oxy state rather than the deoxy state predominantly affects the O₂ affinities. It was concluded that the enhanced O₂ uptake by the picket fence may be due to the stabilization of the oxy state by intramolecular interactions rather than to destabilization of the deoxy state by inhibiting solvation for the active site.     

Measurement of preferential solvation of some glucans in mixed solvent systems by gel-permeation chromatography

Preferential solvation of the glucans amylose, pullulan, and dextran in binary dimethyl-sulfoxide/water (DMSO/H₂O) solvent mixtures has been measured using gel-permeation chromatography. The preferential solvation behavior of the three glucans in DMSO/H₂O solvent mixtures is indistinguishable in the experiments reported. In solvent mixtures with mol ratio DMSO/H₂O less than 1:2, all three glucans are solvated preferentially by H₂O. The maximum extent of preferential solvation by H₂O is about 2.5 mol H₂O/mol of glucose residues. When the DMSO/H₂O mol ratio exceeds 1:2, DMSO solvates the glucans preferentially to a maximum extent of about 1 mol DMSO/mol of glucose residues. An interpretation of the change in preferential solvation with mixed solvent composition is suggested in terms of the known characteristics of the binary solvent system, and the relationship of preferential solvation, reported here, to the absolute solvation of the glucan chains is discussed.     

Continuum solvent studies of the stability of RNA hairpin loops and helices

We apply continuum solvent models to investigate the relative stability of various conformational forms for two RNA sequences, GGAC(UUCG)GUCC and GGUG(UGAA)CACC. In the first part, we compare alternate hairpin conformations to explore the reliability of these models to discriminate between different local conformations. A second part looks at the hairpin-duplex conversion for the UUCG sequence, identifying major contributors to the thermodynamics of a much large scale transition. Structures were taken as snapshots from multi-nanosecond molecular dynamics simulations computed in a consistent fashion using explicit solvent and with long-range electrostatics accounted for using the Particle-Mesh Ewald procedure. The electrostatic contribution to solvation energies were computed using both a finite-difference Poisson-Boltzmann (PB) model and a pairwise Generalized Born model; non-electrostatic contributions were estimated with a surface-area dependent term. To these solvation free energies were added the mean solute internal energies (determined from a molecular mechanics potential) and estimates of the solute entropy (from a harmonic analysis). Consistent with experiment and with earlier solvated molecular dynamics simulations, the UUCG hairpin was found to prefer conformers close to a recent NMR structure determination in preference to those from an earlier NMR study. Similarly, results for the UGAA hairpin favored an NMR-derived structure over that to be expected for a generic GNRA hairpin loop. Experimental free energies are not known for the hairpin/duplex conversion, but must be close to zero since hairpins are seen in solution and duplexes in crystals; out calculations find a value near zero and illustrate the expected interplay of solvation, salt effects and entropy in affecting this equilibrium.     

Application of the local-bulk partitioning and competitive binding models to interpret preferential interactions of glycine betaine and urea with protein surface

Two thermodynamic models have been developed to interpret the preferential accumulation or exclusion of solutes in the vicinity of biopolymer surface and the effects of these solutes on protein processes. The local-bulk partitioning model treats solute (and water) as partitioning between the region at/or near the protein surface (the local domain) and the bulk solution. The solvent exchange model analyzes a 1:1 competition between water and solute molecules for independent surface sites. Here we apply each of these models to interpret thermodynamic data for the interactions of urea and the osmoprotectant glycine betaine (N,N,N-trimethylglycine; GB) with the surface exposed in unfolding the marginally stable lacI HTH DNA binding domain. The partition coefficient K(P) quantifying accumulation of urea at this protein surface (K(P) approximately equal 1.1) is only weakly dependent on urea concentration up to 6 M urea. However, K(P) quantifying exclusion of GB from the vicinity of this protein surface increases from 0.83 (extrapolated to 0 M GB) to 1.0 (indicating that local and bulk GB concentrations are equal) at 4 M GB (activity > 40 M). We interpret the significant concentration dependence of K(P) for GB, predicted to be general for excluded, nonideal solutes such as GB, as a modest (8%) attenuation of the GB concentration dependence of solute nonideality in the local domain relative to that in the bulk solution. Above 4 M, K(P) for the interaction of GB with the surface exposed in protein unfolding is predicted to exceed unity, which explains the maximum in thermal stability observed for RNase and lysozyme at 4 M GB (Santoro, M. M., Liu, Y. F., Khan, S. M. A., Hou, L. X., and Bolen, D. W. (1992) Biochemistry 31, 5278-5283). Both thermodynamic models provide good two-parameter fits to GB and urea data for lacI HTH unfolding over a wide concentration range. The solute partitioning model allows for a full spectrum of attenuation effects in the local domain, encompasses the cases treated by the competitive binding model, and provides a somewhat better two-parameter fit of effects of high GB concentration on lacI HTH stability. Parameters of this fit should be applicable to isothermal and thermal unfolding data for all proteins with similar compositions of surface exposed in unfolding.     

Simulation of Solvation Dynamics in H-Bonding Solvents: Dynamics of Solute–Solvent H-Bonds in Methanol–Water Mixtures

Molecular dynamics (MD) simulations have been undertaken in order to investigate the collective solvent reorganization following an instantaneous electronic charge transfer between distinct atomic sites of diatomic probe molecules immersed in methanol–water mixtures. Our previous studies of solvation dynamics in these mixtures [28,29] are extended here to the analysis of nonequilibrium time-dependent solute–solvent site–site pair distribution functions for the equimolar mixture using two different solute sizes. This has allowed us to obtain a more detailed picture of the solvent reorganization in response to the solute's excitation. Special attention is devoted to the dynamics of rupture and formation of hydrogen bonds between the smaller probe solute and solvent molecules, and its relationship to the molecular mechanisms of solvation dynamics in these systems on distinct time scales. This revised version was published online in June 2006 with corrections to the Cover Date.     

Relationship between preferential interaction of a protein in an aqueous mixed solvent and its solubility

The present paper is devoted to the derivation of a relation between the preferential solvation of a protein in a binary aqueous solution and its solubility. The preferential binding parameter, which is a measure of the preferential solvation (or preferential hydration) is expressed in terms of the derivative of the protein activity coefficient with respect to the water mole fraction, the partial molar volume of protein at infinite dilution and some characteristics of the protein-free mixed solvent. This expression is used as the starting point in the derivation of a relationship between the preferential binding parameter and the solubility of a protein in a binary aqueous solution. The obtained expression is used in two different ways: (1) to produce a simple criterion for the salting-in or salting-out by various cosolvents on the protein solubility in water, (2) to derive equations which predict the solubility of a protein in a binary aqueous solution in terms of the preferential binding parameter. The solubilities of lysozyme in aqueous sodium chloride solutions (pH=4.5 and 7.0), in aqueous sodium acetate (pH=8.3) and in aqueous magnesium chloride (pH=4.1) solutions are predicted in terms of the preferential binding parameter without any adjustable parameter. The results are compared with experiment, and for aqueous sodium chloride mixtures the agreement is excellent, for aqueous sodium acetate and magnesium chloride mixtures the agreement is only satisfactory.     

On the stabilizing action of protein denaturants: acetonitrile effect on stability of lysozyme in aqueous solutions

Stability of hen lysozyme in the presence of acetonitrile (MeCN) at different pH values of the medium was studied by scanning microcalorimetry with a special emphasis on determination of reliable values of the denaturational heat capacity change. It was found that the temperature of denaturation decreases on addition of MeCN. However, the free energy extrapolation showed that below room temperature the thermodynamic stability increases at low concentrations of MeCN in spite of the general destabilizing effect at higher concentrations and temperatures. Charge-induced contribution to this stabilization was shown to be negligible (no pH-dependence was found); therefore, the most probable cause for the phenomenon is an increase of hydrophobic interactions at low temperatures in aqueous solutions containing small amounts of the organic additive. The difference in preferential solvation of native and denatured states of lysozyme was calculated from the stabilization free energy data. It was found that the change in preferential solvation strongly depends on the temperature in the water-rich region. At the higher MeCN content this dependence decreases until, at 0.06 mole fractions of MeCN, the difference in the preferential solvation between native and denatured lysozyme becomes independent of the temperature over a range of 60 K. The importance of taking into account non-ideality of a mixed solution, when analyzing preferential solvation phenomena was emphasized.     

Preferential interaction of dimethyl sulfoxide and phosphatidyl choline membranes

The interaction free energy of dimethyl sulfoxide (DMSO) and two types phospholipid membranes has been assessed from measurements of vapor pressure. The lipids were phosphatidyl cholines with respectively (14:0/14:0) (DMPC) and (16:0/18:1) (POPC) fatty acid chains. The results were expressed in terms of the iso-osmolal preferential interaction parameter, Gamma(mu1), which remained negative under all experimental conditions investigated here. This shows that water-membrane interactions are more favorable than DMSO-membrane interactions. This condition is known as preferential exclusion of DMSO (or preferential hydration of the membrane), and implies that the local (interfacial) concentration of the solute is reduced compared to the bulk. At room temperature and 1 m DMSO, Gamma(mu1) was -0.3 to -0.4 for both lipids. This corresponds to a sizable reduction in the DMSO concentration in a zone including at least the first two hydration layers of the membrane. Possible origins of the preferential exclusion are discussed. As a direct consequence of the pronounced preferential exclusion, DMSO generates an osmotic stress at the membrane interface. This tends to stabilize lipid phases of low surface areas and to withdraw water from multilamellar stacks of membranes. Based on this, we suggest that the preferential exclusion of DMSO explains both the modulation of phase behavior and the constriction of multilamellar aggregates induced by this solute.     

Electrostatic Exclusion of Neutral Solutes from Condensed DNA and Other Charged Phases

Motivated by experiments on condensed DNA phases in binary mixtures of water and a low-dielectric solute, we develop a theory for the electrostatic contribution to solute exclusion from a highly charged phase, within the continuum approximation of the medium. Because the electric field is maximum at the surface of each ion, the electrostatic energy is dominated by the Born energy; interactions between charges are of secondary importance. Neglecting interactions and considering only the competition between the Born energy and the free energy of mixing, we predict that low dielectric solutes are excluded from condensed DNA phases in water-cosolvent mixtures. This suggests that the traditional continuum electrostatic approach of modeling binary mixtures with a uniform dielectric constant needs to be modified. The linking of solute exclusion to solute dielectric properties also suggests a mechanism for predicting the electrostatic contribution to preferential hydration of polar and charged surfaces.     

Atomic-scale analysis of the solvation thermodynamics of hydrophobic hydration.

Molecular dynamics simulations are used to model the transfer thermodynamics of krypton from the gas phase into water. Extra long, nanosecond simulations are required to reduce the statistical uncertainty of the calculated "solvation" enthalpy to an acceptable level. Thermodynamic integration is used to calculate the "solvation" free energy, which together with the enthalpy is used to calculate the "solvation" entropy. A comparison series of simulations are conducted using a single Lennard-Jones sphere model of water to identify the contribution of hydrogen bonding to the thermodynamic quantities. In contrast to the classical "iceberg" model of hydrophobic hydration, the favorable enthalpy change for the transfer process at room temperature is found to be due primarily to the strong van der Waals interaction between the solute and solvent. Although some stabilization of hydrogen bonding does occur in the solvation shell, this is overshadowed by a destabilization due to packing constraints. Similarly, whereas some of the unfavorable change in entropy is attributed to the reduced rotational motion of the solvation shell waters, the major component is due to a decrease in the number of positional arrangements associated with the translational motions.     

Description of hydration free energy density as a function of molecular physical properties

A method to calculate the solvation free energy density (SFED) at any point in the cavity surface or solvent volume surrounding a solute is proposed. In the special case in which the solvent is water, the SFED is referred to as the hydration free energy density (HFED). The HFED is described as a function of some physical properties of the molecules. These properties are represented by simple basis functions. The hydration free energy of a solute was obtained by integrating the HFED over the solvent volume surrounding the solute, using a grid model. Of 34 basis functions that were introduced to describe the HFED, only six contribute significantly to the HFED. These functions are representations of the surface area and volume of the solute, of the polarization and dispersion of the solute, and of two types of electrostatic interactions between the solute and its environment. The HFED is described as a linear combination of these basis functions, evaluated by summing the interaction energy between each atom of the solute with a grid point in the solvent, where each grid point is a representation of a finite volume of the solvent. The linear combination coefficients were determined by minimizing the error between the calculated and experimental hydration free energies of 81 neutral organic molecules that have a variety of functional groups. The calculated hydration free energies agree well with the experimental results. The hydration free energy of any other solute molecule can then be calculated by summing the product of the linear combination coefficients and the basis functions for the solute.     

Sedimentation studies of DNA in concentrated solutions of magnesium salts

The sedimentation coefficient of calf thymus and of T7 DNA was determined at several concentrations up to saturation in solutions of each of the following salts: MgCl₂, MgBr₂, and MgSO₄. Under certain conditions, a plot of the product of the relative viscosity and sedimentation coefficient against the density of the solution has been found to be linear and to extrapolate to zero at a density corresponding to that of the solvated molecule. This behavior was realized in MgSO₄ solutions, the zero intercept occurring at a density of 1.41 g/cc, corresponding to a wafer activity of 0.89. The preferential solvation of DNA in MgSO₄ solutions calculated for this value is 10.5 moles water/mole of nucleotide, in good agreement with published values of solvation of DNA at the same water activity in univalent salt solutions. Since linear plots were not obtained in MgCl₂ and MgBr₂ solutions, buoyant densities could not be determined in these cases. The nonlinear behavior observed in MgCl₂ and MgBr₂ solutions may be due to a change in shape of the DNA molecule at the lower water activities reached in these solutions. The possibility of increased DNA–solute interactions in MgBr₂ and MgCl₂ solutions is also considered as an explanation for the difference in behavior between MgSO₄ and the two magnesium halides.